Thio modified aptamer synthetic methods and compositions

ABSTRACT

The present invention provides a method for concurrent achiral nucleotide modification and amplification using PCR. Provided by this method are NF-kB specific thioaptamers of novel sequence. This invention further provides methods of post-selection aptamer modification wherein one or more selected nucleotides of aptamers of known sequence are substituted with modified achiral nucleotides, particularly achiral thiophosphate nucleotides, wherein the substitution results in increased nuclease resistance while retaining binding efficiency and selectivity. Thiosubstitution of post-selection aptamers with specificity for the nuclear factor, NF-κB, produced in accordance with this method have increased binding affinity and specificity in addition to nuclease resistance. Also provided are methods for fractionating oligonucleotides depending on their degree of thiosubstitution by anion exchange chromatography.

This is a continuation-in-part application claiming priority based onU.S. Provisional Application Ser. No. 60/105,600 filed Oct. 26, 1998.

Work resulting in the present invention was supported in part by UnitedStates Government grants DARPA 9624-107 FP, NIH AI27744, DARPA3-14552-644101 and NIH 3-14546-477999. According, the U.S. Governmenthas certain rights in the invention.

TECHNICAL FIELD OF INVENTION

This invention generally relates the generation of aptamers and to theuse of aptamers as diagnostic and therapeutic agents. More particularly,the present invention relates to methods using combinatorial chemistryto prepare aptamers having controlled thiophosphate replacement in thephosphate backbone for improved binding efficiencies to a target and toRNA and/or DNA products having novel nucleotide sequences and enhancedtarget binding efficacies.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with methods for the preparation of oligonucleotide“decoys”.

Oligonucleotide agents have been shown to have functional activity invitro and thus the promise of therapeutic potential. Some of theseagents are believed to operate via mechanisms such as thesequence-specific antisense translation arrest of mRNA expression orthrough direct binding to protein targets where they function as“decoys”. While oligonucleotide agents show therapeutic promise, variouspharmacological problems must first be overcome.

Oligonucleotide agents have been used as high specificity therapeuticagents in vitro. High sensitivity to nuclease digestion, however, makesoligonucleotide agents unstable and thus impracticable for in vivoadministration by either intravenous or oral routes.

From the foregoing it is apparent the there is a need in the art formethods for generating high binding, nuclease resistant oligonucleotidethat retain their specifity. Also needed are compounds and methods thatpermit the generation of high binding, high specificity, nucleaseresistant oligonucleotide agents that have an improved half-life and aretarget specific.

One target for oligonucleotide decoy targeting is the transcriptionalactivating factor NF-κB, which is activated by many factors thatincrease the inflammatory response. The activation of NF-κB leads to thecoordinated expression of many genes that encode proteins such ascytokines, chemokines, adhesion molecules, etc., which amplify andperpetuate immune response. Because of its pivotal importance in immunefunction, NF-κB has been a desired target for new types ofanti-inflammatory treatments. Current anti-inflammatory treatments suchas glucocorticoids function at least in part through inhibition ofNF-κB. Glucocorticoids, however, have endocrine and metabolic sideeffects when given systematically. Anti-oxidants represent another classof compounds that inhibit NF-κB activation. Currently availableanti-oxidants, however, are relatively weak and have short-term effects.Aspirin and other salicylates also inhibit NF-κB, but only at relativelyhigh concentrations that may have undesirable side effects. Naturallyoccurring inhibitors of NF-κB such as gliotoxin are potent andrelatively specific, but also may have toxic effects.

NF-κB may have a particularly important role in the genesis of endotoxicshock, a disease entity of major clinical importance. In endotoxicshock, a series of intracellular signaling events, in which NF-κBactivation figures importantly, lead to enhanced transcription of avariety of proinflammatory mediator genes, including tumor necrosisfactor α, interleukin-1, inducible nitric oxide synthetase. Thesesecreted mediators in turn lead to increased adhesion moleculeexpression on leukocytes and endothelial cells, increased tissue factorexpression on monocytes and endothelial cells, promoting coagulation,vasodilation, capillary leakiness, and myocardial suppression. (Murphy,et al., New Horizons (1998) 6:181). Strong support for the role of NF-κBin septic shock in humans is afforded by the recent demonstration thatsustained, increased NF-κB binding activity in nuclei of peripheralblood monocytes from septic patients predicted mortality.

From the foregoing it is apparent the there is a need in the art formethods for generating high binding, nuclease resistant aptamers thatretained their specificity.

SUMMARY OF THE INVENTION

Aptamers may be defined as nucleic acid molecules that have beenselected from random or unmodified oligonucleotides (“ODN”) libraries bytheir ability to bind to specific targets or “ligands”. An iterativeprocess of in vitro selection may be used to enrich the library forspecies with high affinity to the target. The process involvesrepetitive cycles of incubation of the library with a desired target,separation of free oligonucleotides from those bound to the target andamplification of the bound ODN subset using the polymerase chainreaction (“PCR”). The penultimate result is a sub-population ofsequences having high affinity for the target. The sub-population maythen be subcloned to sample and preserve the selected DNA sequences.These “lead compounds” are studied in further detail to elucidate themechanism of interaction with the target.

Modulation of the functional attributes of bioactive targets areachieved by aptamer binding. Binding may, for example, interruptprotein•DNA interactions such as those that occur between transcriptionfactors and DNA in the process of gene activation. The ability toeffectively modulate the effects of certain pluripotent transcriptionfactors in vivo would provide a particularly valuable therapeutic tool.

NF-κB is a transcription factor whose activity plays a role in manydisease processes and is thus an potential target for therapeuticcontrol of gene expression. Aptamers can demonstrate high specificity invitro for target proteins and may serve as therapeutics. Highsensitivity to nuclease digestion makes unmodified aptamers unstable incomplex biological systems and tehrefore, unable to mediate the effectsof transcriptional factors such as NF-κB in vivo. Nuclease resistance isparticularly important for the administration of nucleic acid-basedtherapeutics by either intravenous or oral routes. The present inventorsrecognized the need for new concepts in aptamer design that would permitthe generation of effective nuclease resist aptamers and that suchaptamers, if they could be developed, might serve as selective mediatorsof NF-κB activity.

Modification of oligonucleotides such as by thiolation of the phosphoryloxygens of the ODNs can confer nuclease resistance. Thus, it has beenshown by Gorenstein (Farschtschi, N. and Gorenstein, D. G., TetrahedronLett. (1988) 29:6843) and others (See e.g. Nielsen, et al., TetrahedronLett. (1988) 29:291) that sulfur-containing phosphorothioate andphosphorodithioate substituted oligonucleotides show reduced nucleasesusceptibility.

Although phosphoromonothioate analogues ([S]-ODNs) are relativelynuclease resistant, due to the new chiral phosphorus center,phosphoromonothioate mixtures are diasteromeric and thus have variablebiochemical, biophysical and biological properties. Whilestereocontrolled synthesis of P-chiral [S]-ODNs (Yang, et al., J.Bioorganic & Med. Chem. Lett. (1997) 7:2651) represents one possiblesolution to this problem, another lies in the synthesis of modificationsthat are achiral at phosphorus.

In contrast to the phosphomonothioates, the dithioates ([S₂]-ODN)contain an internucleotide phosphodiester group with sulfur substitutedfor both nonlinking phosphoryl oxygens, and are therefore both isostericand isopolar with the normal phosphodiester link. Phosphodithioateanalogues ([S₂]-ODNs) have been synthesized (Gorenstein, et al., U.S.Pat. No. 5,218,088) which have been shown to be highly nucleaseresistant and effective as antisense agents. (Nielsen, et al.,Tetrahedron Lett. (1988) 29:2911; Farschtschi and Gorenstein,Tetrahedron Lett. (1988) 29:6843). In contrast to thephosphoramidite-synthesized monothiophosphate [S]-ODNs, the dithioate[S₂]-ODNs are achiral about the dithiophosphate center, so problemsassociated with diastereomeric mixtures are completely avoided. The[S₂]-ODNs, like the [S]-ODNs, are taken up efficiently by cells.

It has been generally observed that the increased thioation of thephosphoryl oxygens of ODNs often leads to their enhanced binding tonumerous proteins. For example, single-stranded [S₂]-ODNs are 36-600times more effective in inhibiting HIV reverse transcriptase than normalantisense ODN or the [S]-ODN (Caruthers, M. H., Abstract, InOligonucleotides As Antisense Inhibitors of Gene Expression, RockvilleMd., Jun. 18-21, 1989).

It has also been noted, however, that oligonucleotides possessing highmonothio- or dithio-phosphate backbone substitutions appear to be“stickier” towards proteins than normal phosphate esters, possibly basedon the charge characteristics of the sulfonated nucleotides. Theincreased stickiness of thiolated ODNs results in loss of specificity,thus, defeating the promise of specific targeting offered by aptamertechnology. Loss of specificity is critical in DNA binding proteins-DNAinteractions, because most of the direct contacts between the proteinsand their DNA binding sites are to the phosphate groups. As a furthercomplication, it has been found that certain thiosubstitution can leadto structural perturbations in the structure of the duplex (Cho, et al.,J. Biomol. Struct. Dyn. (1993) 11, 685-702).

In one embodiment of the present invention provides a novel applicationof DNA polymerase to incorporate chiral phosphorothioates and replicatea random sequence library simultaneously in order to prepare achiralNF-κB specific aptamers. A random phosphodiester oligonucleotidecombinatorial library is synthesized wherein constituentoligonucleotides comprise at least a set of 5′ and 3′ PCR primernucleotide sequences flanking a randomized nucleotide sequence. Thelibrary is amplified enzymatically using a mix of four nucleotidesubstrates, wherein at least a portion of the total quantity of at leastone but no more than three of the nucleotides is modified, to form amodified oligonucleotide combinatorial library.

Furthermore, only a portion of the total quantity of given a nucleotidebase may be modified and/or more than one modified nucleotide base isincluded in the amplification mix, thereby increasing the number ofpotential substitution. The modified oligonucleotide combinatoriallibrary is next contacted or mixed with a target protein, e.g., an NF-kBdimer or constituent subunit protein, and the subset of oligonucleotidesbinding to the protein is isolated. The subset of NF-kB bindingoligonucleotides is again amplified enzymatically using the mix of fournucleotide substrates, including modified nucleotides to form a modifiedoligonucleotide sub-library. The amplification and isolation steps arerepeated iteratively until at least one aptamer having one or moremodified oligonucleotides of defined sequence is obtained.

The unique chemical diversity of the oligonucleotide libraries generatedby methodologies provided herein stems from both the nucleotidebase-sequence and phosphorothioate backbone sequence. The present methodprovides achiral oligonucleotide products whether the amplificationsubstrates are monothiophosphates or dithiophosphates. The presentthioaptamer methodology provides compounds that are an improvement overexisting antisense or “decoy” oligonucleotides because of theirstereochemical purity. Chemically synthesized phosphorothioates may be adiastereomeric mixture with 2^(n) stereoisomers with n being the numberof nucleotides in the molecule. These preparations are unsuitable foruse in humans because only a small fraction of the stereoisomers willhave useful activity and the remaining could have potential adverseeffects. In contrast, enzymatically synthesized oligonucleotides arestereochemically pure due to the chirality of polymerase active sites.Inversion of configuration is believed to proceed from R_(p) to S_(p)during incorporation of dNMPαS into the DNA chain. The presentdithiophosphate aptamers are free from diastereomeric mixtures.

The present inventors recognized that it is not possible to simplyreplace thiophosphates in a sequence that was selected for binding witha normal phosphate ester backbone oligonucleotide. Simple substitutionwas not practicable because the thiophosphates can significantlydecrease (or increase) the specificity and/or affinity of the selectedligand for the target. It was also recognized that thiosubstitutionleads to a dramatic change in the structure of the aptamer and hencealter its overall binding affinity. The sequences that were thioselectedaccording to the present methodology, using as examples of DNA bindingproteins both NF-IL6 and NF-κB, were different from those obtained bynormal phosphate ester combinatorial selection.

The present invention takes advantage of the “stickiness” of thio- anddithio-phosphate ODN agents to enhance the affinity and specificity to aprotein target. In a significant improvement over existing technology,the method of selection concurrently controls and optimizes the totalnumber of thiolated phosphates to decrease non-specific binding tonon-target proteins and to enhance only the specific favorableinteractions with the target. The present invention permits control overthat phosphates to be thio-substituted in a specific DNA sequence,thereby permitting the selective development of aptamers that have thecombined attributes of affinity, specificity and nuclease resistance.

In one embodiment of the present invention, a method of post-selectionaptamer modification is provided in which the therapeutic potential ofthe aptamer is improved by selective substitution of modifiednucleotides into the aptamer oligonucleotide sequence. A target bindingaptamer is identified and the nucleotide base sequence determined.Modified achiral nucleotides are substituted for one or more selectednucleotides in the sequence. In one embodiment, the substitution isobtained by chemical synthesis using dithiophosphate nucleotides. Theresulting aptamers have the same nucleotide base sequence as theoriginal aptamer but by virtue of the inclusion of modified nucleotidesinto selected locations in the sequences improved nuclease resistance isobtained.

Using the method disclosed hereinbelow, a family of aptamers withmodifications at different locations was created and the bindingefficiency for the target determined. Using the disclosed method,specific NF-κB binding aptamers were created that are not only morenuclease resistant but have increasing binding affinity over unmodifiedaptamers of the same sequence. In contrast to fully thiolated aptamersof the same sequence, the selectively thiolated aptamers of the presentinvention had greater selectivity for the desired target NF-κB dimers.

In one embodiment of the present invention, a process for fractionatingoligonucleotides with varying degrees of thioation is provided. A crudeoligonucleotide mixture is dissolved in a starting solvent, e.g., water.The dissolved oligonucleotide is loaded onto an anion-exchange column,e.g., an FPLC of HPLC Mono Q column. Oligonucleotides with varyingdegrees of thioation are sequentially eluted with a buffered saltgradient. In addition to the fractionation and collection of specificthiolation species, the method permits the ready removal of undesiredmonothiophosphate contaminates.

The controlled thiolation methodology of the present invention isapplicable to the design of specific, nuclease resistant aptamers tovirtually any target including without limitation amino acids, peptides,polypeptides (proteins), glycoproteins, carbohydrates, nucleotides andderivatives thereof, cofactors, antibiotics, toxins, and small organicmolecules including inter alia, dyes, theophylline, and dopamine. It iscontemplated, and within the scope of this invention, that the instantthioaptamers encompass further modifications to increase stability andspecificity including for example disulfide crosslinking. It is furthercontemplated and within the scope of this invention that the instantthioaptamers encompass further modifications including, e.g.,radiolabeling and/or conjugation with reporter groups, such as biotin orfluorescein, or other functional groups for use in in vitro and in vivodiagnostics and therapeutics.

The present invention further provides the application of thismethodology to the generation of novel thiolated aptamers specific fornuclear factors such as, e.g., NF-IL6 and NF-κB. The NF-κB/Reltranscription factors are key mediators of immune and acute phaseresponses, apoptosis, cell proliferation and differentiation. TheNF-κB/Rel transcription factors are also key transactivators acting on amultitude of human and pathogen genes, including HIV-1.

Several family members of NF-κB/Rel have been identified based not onlyon sequence, but also structural and functional homology. Members ofthis protein family are divided into two groups based on differences instructures, functions and modes of synthesis: one group includes of theprecursor proteins p105 and p100 with ankyrin repeat domains in theircarboxyl termini. Proteolytic processing removes the carboxyl halves toyield the mature forms p50 and p52, respectively. The subsequenthomodimers are weak transcriptional activators at best, since they lackcarboxyl transactivation domains. A second group, including p65 (RelA),c-Rel, v-Rel, Rel B, Dorsal, and Dif are not synthesized as precursorsand are sequestered in the cytoplasm by association with inhibitors(IκB) or precursor proteins (p100 and p105). Homo- or hetero-dimersincluding at least one member from this group are strong transcriptionalactivators.

Both groups of NF-κB/Rel proteins can form homo- and hetero-dimers.Hetero-dimers consisting of p50 and p65 (RelA) are the ubiquitouslyexpressed form of the NF-κB transcription factor. Upon nuclear entry,NF-κB/Rel proteins bind to specific sites resembling the consensussequence, GGGRNNT(Y)CC. These sites are found in promoters and enhancersof a variety of cellular genes including genes involved in the immuneresponse (IgκB, IL-2, IL-2Rα, cyclooxygenase-2), acute phase responsegenes (TNFα, IL-1, IL-6, TNFβ), viruses (HIV, CMV, SV-40), growthcontrol proteins (p53, c-myc, ras, GM-CSF), NF-κB/Rel and IκB proteinsand cell adhesion molecules (1-CAM, V-CAM and E-selectin) and many othergenes. The affinity of the NF-κB/Rel proteins for DNA is determined bythe sequence of the binding site. Different combinations of NF-κB/Relproteins in dimers influence binding site preferences and affinities.Therefore, it is likely that different forms of NF-κB activate differentsets of target genes with respect to certain NF-κB binding sites.

The present invention provides an aptamer capable of discriminatingbetween, and binding to, a single NF-κB/Rel protein dimer species. Priorart backbone aptamers or target site sequences or fullymonothiophosphate sequences bind to multiple proteins in cell culture.The present invention provides for both NF-IL6 and NF-κB, aptamers thatare long enough to permit interaction with multiple proteins andprovides methods to specifically select multi-protein complexes and todiscriminate among the different transcription factors.

The present structure-based dithiophosphate and combinatorialmonothiophosphate selection system provides for the identification ofaptamers that have high specificity, and high affinity for DNA bindingproteins, e.g, a single NF-κB heterodimer, in a cellular extract. Thepresent invention encompasses the development of separate aptamerstargeting any one of the 15 possible combinations of 5 homo- andhetero-dimers of the 5 different forms of NF-κB/Rel.

The present invention discloses the use of NF-κB dithioate aptamers toselectively bind various NF-κB hetero- and homo-dimers to down-regulatethe pathogenic aspects of systemic inflammation and/or up-regulate theprotective/anti-inflammatory aspects of the response and thus to protectagainst endotoxic shock and LPS tolerance.

NF-κB is activated by many factors that increase the immune response.NF-κB activation leads to the coordinated expression of many genes thatencode proteins such as cytokines, chemokines, adhesion molecules, etc.all amplifying and perpetuating the immune response. In addition thereis evidence that X-rays (used in treatment of Kaposi's sarcoma) arepotent inducers of NF-κB, triggering HIV proviral transcription. (Faure,et al., AIDS Research & Human Retroviruses (1996) 12, 1519-1527). TheNF-κB specific thioaptamers of the present invention provide for ageneration of anti-AIDS therapeutics with specific application duringthe treatment of patients with Kaposi's sarcoma.

Alternatively, the present invention discloses the use of NF-κB specificthioaptamers targeted to p50•p50 or p52•p52 (inhibitors of NF-κBtransactivation) to activate κB-specific gene expression (Zhang, et al.,Blood (1998) 91:4136) and aid in “smoking out” latent reservoirs of HIVby inducing expression of latent virus infected cells that are thensusceptible to combination anti-viral therapy.

The NF-κB aptamers of the present invention have utility in the studyand treatment of the many diseases in which this transcription factorplays a critical role in gene activation, especially acute phaseresponse and inflammatory response. These diseases include (but are notlimited to): bacterial pathogenesis (toxic shock, sepsis), rheumatoidarthritis, Crohn's disease, generalized inflammatory bowel disease,asbestos lung diseases, Hodgkin's disease, prostrate cancer, ventilatorinduced lung injury, general cancer, AIDS, human cutaneous T celllymphoma, lymphoid malignancies, HTLV-1 induced adult T-cell leukemia,atherosclerosis, cytomegalovirus, herpes simplex virus, JCV, SV-40,rhinovirus, influenza, neurological disorders, and lymphomas.

Another aspect of the present invention is to both thioselect and designaptamers (monothiophosphate and dithiophosphate, as well as otherbackbone substitutions) that specifically target protein•proteincomplexes such as the “enhanceosome”. As part of the present invention,enhanced aptamer selectivity and binding has been achieved respective toprotein protein contacts as well as protein•aptamer contacts. Thiolatedaptamers allow the formation of a specific protein•protein•aptamercomplex capable of preferentially forming an inactive enhanceosome on agene that is unable to interact with the basal transcriptional factors.Using the disclosed method and compositions, aptamers may be designed orselected that are specific for the multiprotein enhanceosome complex butnot for the complete transcriptional activation complex.

The aptamers themselves have utility as biochemical research tools ormedical diagnostics agents in cell culture, animal systems, in vitrosystems and even to facilitate hot start PCR through the inhibition ofhigh temperature polymerases. Three dimensional structural determinationof modified aptamers with both high binding efficiency and specificityaccording to the present invention also provides a vehicle for drugdesign structural modeling of the active sites of desired drug targets.

The invention contemplates the use of PCR to incorporate up to threedNTPαSs into DNA. Incorporation of dNTPαSs is important because greatersubstitution may impart greater nuclease resistance to the thiolatedaptamers. The use of dNTPαSs is also important because the initiallibrary will also have greater diversity. Using the present inventionthiolated aptamers may be selected having one or more thio-modifiednucelotide substitutions.

Single-stranded nucleic acids are also known to exhibit uniquestructures. The best documented single-stranded nucleic acids structuresare of single-stranded RNA. Single-stranded DNA can also adopt uniquestructures. The present invention is applicable to the selection ofsingle-stranded phosphorothioate aptamers comprised of either RNA orDNA. Such single-stranded aptamers are applicable to both DNA (i.e.,cell surface receptors, cytokines, etc.) and non-DNA binding proteins.

It is contemplated that the present methods and procedures may bescaled-up as would be necessary for high throughput thioaptamerscreening and selection. For example, 96 well microtiter plates may beused to select aptamers to a number of different proteins under numerousconditions.

This technology is directly applicable to protein/DNA chip technologyaccording to methodologies, e.g., those described in U.S. Pat. No.5,874,219 incorporated herein by reference. By attaching the highlyselective aptamers to a support, protein/DNA chip permits theidentification and quantitation of the protein levels of all possibleforms of not only NF-κB/Rel proteins but many other transcriptionfactors and other proteins that function by forming differentprotein•protein complexes (i.e. NF-IL6/Lip/NF-κB,Bad/Bax/BCL-X_(S)/BCL-X_(L), etc.). A 2D-arrayed chip would be capableof discriminating among 100's or even 1000's of protein•proteincomplexes in the cell simultaneously. The present invention disclosesthe attachment of nucleic acids, rather than unstable proteins, to thechip substrates, permitting current DNA chip technologies(photolithography, ink jet, etc.) may be used. Solid state chiptechnology provides structure-based and combinatorial drug designprogram as well as general medical diagnostics, making it feasible tomonitor the varying populations of different protein-protein complexesresulting from disease progression or drug treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, includingfeatures and advantages, reference is now made to the detaileddescription of the invention along with the accompanying figures:

FIG. 1 depicts a competitive fluorescence polarization titration of5′-labeled fluorescein-20-mer duplex/NF-IL6 TCD dimer complex;

FIG. 2 depicts relative sensitivity of Family A 66-mers to degradationby DNase I. Unmodified, phosphoryl duplex (●) and monothiophosphorylatedat non-primer dA sites only (□);

FIG. 3 is a negative control, at a concentration of up to 4 μm,thiophosphate clone 98 (Family A) was shown not to bind to anothertranscription factor, NF-κB (p65 dimer);

FIG. 4 depicts these titrations performed directly whereby each aptamerwas fluorescein labeled and the protein was titrated into the solution;

FIG. 5 shows results of a competition assay for binding CK-1 42-meraptamers;

FIG. 6 is a graph showing the inhibition of p65 homodimer binding by[S₂]-ODN (XBYs);

FIG. 7 a shows the sequences of thioselected NF-kB aptamers according tothe present invention while;

FIG. 7 b shows a consensus sequence determined therefrom;

FIG. 8 shows ³¹P NMR spectrum of thymidine 3′-O-phosphorodithioatesingle stranded sequence 5 (SEQ ID NO.: 43) recorded on a Varian Unityplus spectrometer operating at 242 MHZ;

FIG. 9 shows inhibition of p65 homodimer binding by thymidine3′-O-phosphorodithioate duplex aptamers 6-11 (SEQ ID NOS.: 34-39);

FIG. 10 a shows a diagrammatic representation of the sites ofthiophosphate and dithiophosphate modification of the nucleotidebackbone;

FIG. 10 b shows a diagrammatic representation of thymidine3′-O-phoshorodithiophosphate;

FIG. 11 a shows the dose response curve of LPS administration on serumTNF in a guinea pig model; and

FIG. 11 b shows the in vivo effect of a NF-kB specific thioaptamer onreducing serum TNF production in response to LPS challenge.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts whichcan be employed in a wide variety of specific contexts. The specificembodiment discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

Abbreviations

The following abbreviations are used throughout this application:

-   -   bZIP—basic leucine zipper    -   BSA—bovine serum albumin    -   CD—circular dichroism    -   C/EBPβ—CCAAT-enhancer binding protein β    -   DNase 1—Deoxyribonuclease 1    -   DTT—dithiothreitol    -   EDTA—ethylene diamine tetraacetic acid    -   I16—Interleukin-6    -   kb—kilobase (pairs)    -   kD—kilodalton    -   K_(obs)—observed binding constant        Dithiophosphates are substituted at both oxygens and are thus        achiral. Phosphorothioate nucleotides are commercially available        or can be synthesized by several different methods known in the        art.

“Modified” means oligonucleotides or libraries comprisingoligonucleotides wherein one or more of the four constituent nucleotidebases are analogues or esters of nucleotides normally comprising DNA orRNA backbones and wherein such modification confers increased nucleaseresistance. Thiophosphosphate nucleotides are an example of modifiednucleotides.

“Phosphodiester oligonucleotide” means a chemically normal (unmodified)RNA or DNA oligonucleotide.

Amplifying “enzymatically” refers to duplication of the oligonucleotideusing a nucleotide polymerase enzyme such as DNA or RNA polymerase.Where amplification employs repetitive cycles of duplication such asusing the “polymerase chain reaction”, the polymerase is a heat stablepolymerase such as the DNA polymerase of Thermus aquaticus.

“Contacting” in the context of target selection means incubating aoligonucleotide library with target molecules.

“Target molecule” means any molecule to which specific aptamer selectionis desired.

“Essentially homologous” means containing at least either the identifiedsequence or the identified sequence with one nucleotide substitution.

“Isolating” in the context of target selection means separation ofoligonucleotide/target complexes, preferably DNA/protein complexes,under conditions in which weak binding oligonucleotides are eliminated.In one preferred embodiment DNA/protein complexes are retained on afilter through which non-binding oligonucleotides are washed.

By “split synthesis” it is meant that each unique member of thecombinatorial library is attached to a separate support bead on a twocolumn DNA synthesizer, a different thiophosphoramidite is first addedonto both identical supports (at the appropriate sequence position) oneach column. After the normal cycle of oxidation and blocking (whichintroduces the dithiophosphate linkage at this position), the supportbeads are removed from the columns, mixed together and the mixturereintroduced into both columns. Synthesis may proceed with furtheriterations of mixing or with distinct nucleotide addition.

A recent advance in combinatorial chemistry has been the ability toconstruct and screen large random sequence nucleic acid libraries foraffinity to proteins (Gold et al., Proc. Natl. Acad. Sci. U.S.A. (1997)94: 59; Tian et al., RNA (1995) 1: 317; Ekland et al., Science (1995)269: 364). The nucleic acid libraries are usually selected by incubatingthe target protein with the library and then employing a method ofseparating the non-binding species from the bound. The bound fractionsare then amplified using PCR and subsequently incubated again with theprotein for a second round of the screening or selection process. Theseiterations are repeated until the library is enhanced for sequenceshaving high affinity for the target protein.

Agents selected from combinatorial libraries of RNA and DNA in the pasthave normal phosphate ester backbones and thus are generally unsuitableas drugs in vivo because of their nuclease susceptibility. Althoughvarying degrees of nuclease resistance may be obtained using modifiednucleotides, for example, by thiosubstitution at the non-binding oxygengroups of the phosphate backbone, the present inventors recognized thatthe functional effect of substitution of nuclease resistantthiophosphates could not be predicted since the sulfur substitution maylead to either decreased or increased binding to a specific protein.

The present inventors developed a novel combinatorial approach involvingthe construction and screening of a phosphorothioate DNA library. In oneembodiment, the target selected was the nuclear factor for IL6 (NF-IL6),a basic leucine zipper transcription factor involved in the induction ofacute-phase responsive and cytokine gene promoters in response toinflammation (Akira & Kishimoto, Immun. Rev. (1992) 127:25).

The following examples are include for the sake of completeness ofdisclosure and to illustrate the methods of making the compositions andcomposites of the present invention as well as to present certaincharacteristics of the compositions. In no way are these examplesintended to limit the scope or teaching of this disclosure.

EXAMPLE 1 Thioselection of Phosphorodithioate Aptamers Binding to NF-IL6

The present invention provides oligonucleotide combinatorial methodsthat may be extended to selection not only of base sequence but ofphosphate (or monothiophosphate) backbones as well. The bestmonothiophosphate aptamers were obtained using the following method. Asan example, binding was increased at least 5-fold to the NF-IL6 trypticcore domain (TCD) than the normal backbone sequence. The sequencesselected, while related to the normal backbone CAAC/T half-sites (forFamily B below), show distinct differences that are likely attributableto alterations in the nature of the protein-phosphate backboneinteractions in the complex.

Because Taq polymerase can use up to 3 different dNTP(αS)s in thepolymerization reaction, further backbone substitutions are possible.The present invention contemplates the incorporation of bothtriphosphate and triphosphate(αS) nucleotides in the PCR mix so that alibrary of both phosphate and monothiophosphate backbones may berandomized at the same base position, greatly increasing the diversityof the library.

A. Library Generation

A random combinatorial library of normal phosphoryl backboneoligonucleotides was synthesized by automated DNA synthesis (MidlandCertified Reagents, Midland, Tex.) programmed to include all 4 monomerbases of the oligonucleotide during the coupling of residues in arandomized segment. This synthetic library has PCR primer segments atthe 5′ and 3′ ends flanking the randomized region and thus can bereplicated and amplified by Taq DNA polymerase (AMPLITAQ, Perkin Elmer).A 66-mer has been used with a 22 base pair random central segmentflanked by 21 and 23 base pair PCR primer regions: (SEQ ID NO.:1)5′CAGTGCTCTAGAGGATCCGTGAC N₂₂ CGAAGCTTATCGATCCGAGCG3′

The resulting library thus exists as a population with potentially 4²²(10¹³) different sequences. The oligonucleotide library withphosphorothioate backbone substituted at dA positions was thensynthesized by PCR amplification of the 66-mer template usingcommercially available Taq polymerase and a mix of dATP(αS), dTTP, dGTPand dCTP as substrates (Pharmacia, Inc.). The PCR amplification of thestarting random library included: 40 μM each of dATP(αS), dTTP, dGTP anddCTP, 500 μM MgCl₂, 2.9 μM 66mer random template, 5 U Taq polymerase and400 nM each primer in a total volume of 100 μL. PCR was run for 25cycles of 95° C./1 min, 72° C./1 min. This polymerase is known to PCRamplify a phosphorothioate backbone template (Nakamaye et al. Nucl.Acids Res. (1988) 16: 9947) so long as the dNTP(αS)'s are limited to nomore than 3 different bases in the mixture (Ciafre et al. Nucl. AcidsRes. (1995) 23:4134). It also acts stereospecifically to incorporate theS_(p)-diastereomers of dNTP(αS)'s and is believed to yield the R_(p)stereoisomer as is found for other polymerases (Eckstein, F. Ann. Rev.Biochem. (1985) 54: 367).

B. NF-IL6 Preparation

When full length NF-IL6 complexed with DNA is exposed to trypsin, a 9.5kDa fragment is identified as the smallest fragment stably resistant toproteolysis. This basic leucine zipper (bZIP) domain peptide spans aminoacids A²⁶⁶-C³⁴⁵, and is termed the NF-IL6 tryptic core domain (TCD).

High level expression of recombinant NF-IL6 bZIP region in E. coli wasachieved in the T7 promoter/polymerase system. The TCD expressed as anonfusion protein constitutes 30% of the total soluble E. coli proteinand was purified as previously described (Brasier, et al., J. Biol.Chem. (1994) 269: 1034). The TCD bZIP domain binds DNA in a mannerindistinguishable from full length NF-IL6. Electrospray massspectrometry indicates that the mass of TCD is 18,926 Da. These dataindicated that TCD is a covalently linked dimer through its C-terminaldisulfide bond. The present selection, however, was performed underconditions in which the disulfides are reduced and the TCD exists as anon-covalent dimer.

C. NF-IL6 Thiophosphate Selection

The random library was screened to determine sequences that haveaffinity to the bZIP domain of NF-IL6. PCR amplification of the singlestranded library provided chiral duplex phosphorothioate 66-mer at alldA positions (except for the primer segments). A filter-binding methodwas used for enrichment of binding sites, although other methods knownin the art are also suitable. The PCR amplified random library of thechiral duplex phosphorothioate 66-mer at dA positions (100 pmols) wasincubated with 6.6 pmols TCD in 50 μl buffer containing 10 mM Tris, pH7.5, 1 mM DTT and 50 to 400 mM KCl and filtered through Millipore HAWP25mm nitrocellulose filters (following a modification of the protocol fromThiesen et al., Nucl. Acids Res. (1990) 18: 3203). The filters had beenpreviously presoaked in 1× binding buffer that contains no protein orDNA (10 mM Tris, pH 7.5, 1 mM DTT and 50 to 400 mM KCl). Under theseconditions the DNA/protein complexes were retained on the filter. Thefilter was then washed with 10 ml of 1× binding buffer to remove themajority of the DNA that only weakly bound to the protein.

A 1 ml solution of 8 M urea and 4 M NaCl was then added to elute theprotein-bound DNA. A negative control without protein was performedsimultaneously to monitor any non-specific binding of the thiophosphateDNA library to the nitrocellulose filter. DNA was ethanol precipitatedand once again PCR amplified with the dATP(αS) nucleotide mix. The PCRthermal profile was different than that used to make the startinglibrary: 95° C./1 min, 55° C./1 min, 68° C./1 min for 25 cycles. The PCRproducts were analyzed by 15% non-denaturing polyacrylamide gelelectrophoresis.

At various stages of the selection process the resulting libraries werecloned and plasmids from individual colonies sequenced. The normalphosphate ester 66-mer duplexes in the libraries were sub-cloned usingthe TA cloning kit (Invitrogen). As a control, four clones were alsosequenced from the original combinatorial library and shown to haverandom sequence.

The present invention provides a thiophosphate backbone combinatoriallibrary created by PCR methods with substitution of appropriate dNTP(αS)in the Taq polymerization step. This combinatorial thiophosphate duplexlibrary was successfully screened for binding to the TCD of NF-IL6 by afilter binding method that was modified to minimize non-specific bindingof the thiophosphate oligonucleotides to the nitrocellulose filter. Thethiophosphate substituted DNA may be eluted from the filter using, e.g.,high salt, protein denaturing conditions described or other conditionsknown in the art. Subsequent ethanol precipitation and another PCRthiophosphate amplification provide product pools for additional roundsof selection may also be used to further select for high affinitybinding. In order to increase the stringency of binding of the remainingpool of DNA in the library (thereby, selecting tighter binding membersof the library), the KCl concentration was increased in subsequentrounds from 50 to 400 mM. The stringency of selection was alsomanipulated by lowering the amount of protein as the iteration numberincreased.

The first selection was carried through 7 iterations. Only 3 clones wereselected and sequenced (Table-1) at this stage of the selection process.In all 3 early round clones (3:3), a general consensus sequence wasfound with a stretch of 8-11 A/C's including the sequences: ACAACCC orACACCACC. NF-IL6 is a CCAAT/enhancer-binding protein (C/EBPβ) withspecificity for two CCAAC/T boxes. Thus in these early rounds ofselection, the thiophosphate substitution at dA did not dramaticallyaltered the affinity for the “CAAC”-like box.

A second independent selection included 10 iterations and yielding thesequences also shown in Table 1 (4 of 4 clones). As shown in Table 1,the two independent selection studies identified a single uniquesequence (compare clones #2 and #7). While the ACAACCC sequence onceagain appeared (#7), another unique new sequence (dGGGCCC GCTGT ACATG CACACG, SEQ ID No.: 5, clones #4-6) was found for the entire 22-bprandomized segment. The Table has been divided to emphasize homologyamong 5-6 bp consensus, putative recognition units: “GC-box” Duplexformat “5′ACAGC.GCTGT” 5′ ACAGC    TGTCG 5′ “5′ACATG.CATGT” 5′ ACATG   TGTAC 5′ “5′ACACG.CGTGT” 5′ ACACG    TGTGC 5′

Nascent elements of this new variation were also observed at round 7 ofthe first selection(GC box, ACA and ACACG units). TABLE I Sequences ofVariable 22-mer Region in 66-mer Thiophosphate Aptamers, Selected afterIndicated Rounds.* CONSENSUS GC-box ACAGC.GCTGT ACATG.CATGT ACACG.CGTGT1st INDEPENDENT SELECTION STUDY, ROUND 7  1 (SEQ ID No.:2) 5′d GCC GTCCACATA C G ACACCACC  2 (SEQ ID No.:3) 5′dGGCC GACCGC ACA G C ACAACCC  3(SEQ ID No.:4) 5′dGGC GCGGAT ACAAC C C ACACGC 2nd INDEPENDENT SELECTIONSTUDY, ROUND 10  4 (SEQ ID No.:5) 5′dGGGCCC GCTGT ACATG C ACACG  5 (SEQID No.:5) 5′dGGGCCC GCTGT ACATG C ACACG  6 (SEQ ID No.:5) 5′dGGGCCCGCTGT ACATG C ACACG  7 (SEQ ID No.:6) 5′dGGCC GACCGC ACA G C ACAACCCROUND 16: Family A  8 (SEQ ID No.:7) 5′GGGCCC GCTGT ACATG C ACACG  9(SEQ ID No.:7) 5′GGGCCC GCTGT ACATG C ACACG 10 (SEQ ID No.:7) 5′GGGCCCGCTGT ACATG C ACACG 11 (SEQ ID No.:7) 5′GGGCCC GCTGC ACGTG C ACACG 12(SEQ ID No.:8) 5′GGGCCC GCTGT ACACG C ACACG ROUND 16: Family B 13 (SEQID No.:9) 5′ CCC GTTGT TGTCCCACT CCACG 14 (SEQ ID No.:10) 5′ CCC GTTGTTGTCCCGCT CCACG*Sequences are aligned to highlight the consensus elements (underlined).All sequences are written such that the first six flanking 5′ and3′ primer sequences are all 5′GCTTCG and 5′CTCACC, respectively.

The 10th pool of the second selection was carried through an additional6 iterations and in the 7 clones sequenced, two major sequence familieswere obtained (Table 1): Family A) typified by 5′dGGGCCC GCTGT ACATG CACACG (SEQ ID No.: 7) and Family B) typified by 5′dCCC GTTGT TGTCCCACTCCACG (SEQ ID No.: 9). Within these 22-base sequences only 1 or 2 basechanges were found for each family (3 of the 7 were identical sequences;clones #8-10). Note that even by round 10, three members of the 22-basesequence are identical to the A family consensus sequence (clones #4-6).Family B retains the early round CAAC.GTTG consensus while family A haslost all “traditional” C/EBPβ CAAC/T box sequence homology. Anadditional group of 25 clones were sequenced (data not shown) and the22-mers were found to also fall within the two families (identical tothe consensus sequence or differing by only 1 nucleotide).

These results differ from normal phosphate ester backbone in vitroselection studies with NF-IL6, where a traditional CAAC box wasidentified using the same TCD of NF-IL6 and 66-mer library underidentical selection conditions. Osada, et al. (J. Biol. Chem. (1996)271:3891), used full-length C/EBPβ and a 16 nucleotide randomizedlibrary to determine a 10-bp consensus sequence showing the expected twohalf-site GTTGC.GCAAC in a palindromic sequence as shown in Table 2.TABLE 2 Comparison of Putative Phosphodiester and ThiophosphateConsensus Recognition Sequences Consensus phosphate GTTGC GCAAC (SEQ IDNo.: 11) ester (Osaka) Consensus GCTGT ACATG (SEQ ID No.: 12)thiophosphate ester (Family A) Consensus GTTGT CCCAC (SEQ ID No.: 13)thiophosphate ester or (Family B) GTTGT TGTCC* (SEQ ID No.: 14)*(alignment of a consensus sequence is more difficult for Family Bmembers)

Thiophosphate substitution of dA altered the sequence selected, therebyeliminating the sequential AA consensus sequence (Family A only) foundin all other phosphate selection studies.

In both normal phosphate and thiophosphate duplex 10-mers, 4-5 dA's maybe found, indicating that thiophosphate substitution for the dA residueshas not had a deleterious effect on binding.

D. Affinity Measurements by Fluorescence Polarization

The affinity of the selected oligo's or libraries have been measured byfluorescence anisotropy (Heyduk et al. Proc. Natl. Acad. Sci., U.S.A.(1990) 87: 1744). Fluorescence polarization titrations using increasingconcentrations of the recombinant protein to bind a palindromic5′-labeled fluorescein C/EBPβ 20-mer binding site with a normalphosphate ester backbone (dTGCAGATTGCGCAATCTGCA: SEQ ID NO.: 15) gave anobserved binding constant, K_(obs), of 10 nM.

Thiophosphate 66-mers were PCR amplified, phenol extracted and ethanolprecipitated. DNA purity was >95% as assessed by PAGE gels. Varyingconcentrations of 66-mers, 5′-Labeled fluorescein-20-mer palindromicbinding site and NF-IL6 TCD dimer were incubated in 10 mM Tris, pH 7.5,50 mM KCl, 1.0 mM DTT buffer for 1 hour prior to fluorescencepolarization measurements. Concentrations of 66-mer were calculated at20 OD₂₆₀/mg. The observed binding constant, K_(obs), represented the66-mer concentration providing a 50% decrease in the fluorescencepolarization intensity change. Fluorescence polarization titrations werecarried out on a Panvera Beacon polarimeter.

The monothiophosphate libraries and individual 66-mer sequences wereused as competitors to dissociate a fluoroscein-labeled, normal backboneduplex C/EBPβ 20-mer bound to the bZIP protein. As shown in FIG. 1, anindividual monothiophosphate 66-mer cloned from the 10th selection round(clone #7) gave a K_(obs) of <2 nM. DTT and 50 to 400 mM KCl).

FIG. 2 depicts relative sensitivity of Family A 66-mers to degradationby DNase I. Unmodified, phosphoryl duplex (●) and monothiophosphorylatedat non-primer dA sites only (□).

Following a similar competitive titration, monothiophosphate (at dA)clones #8 and 13 (consensus Family A and B, respectively) also gave a Kbof <2 nM (FIG. 3). As a negative control (data not shown), at aconcentration of up to 4 μM, thiophosphate clone #8 (Family A) was shownnot to bind to another transcription factor, NF-κB (p65 dimer).

These titrations were also performed directly where each aptamer wasfluorescein labeled and the protein was titrated into the solution.These assays gave similar estimates of the binding constants (Family A 5nM and Family B 3 nM, monomer concentration giving 50% saturation, FIG.4).

Stoichiometric titration of 66-mers with TCD established that laterrounds (FIG. 1) bound the protein dimer with an approximate 2:1 (proteindimer: DNA duplex) stoichiometry. Initial rounds bound to TCD with a 1:1stoichiometry (FIG. 1). This change in stoichiometry may explain theremarkable selection of a full 22-base sequence when the NF-IL6consensus site is believed to be only 10 bases in length.

The crystal structure of leucine zipper proteins such as GCN4 and AP-1in the DNA cocrystal are coiled coils (Ellenberger, et al., Cell (1992)71: 1223), with the basic region coiling into a helix to bind in themajor groove in each of the half sites. In a bZIP protein such asNF-IL6, it would be expected that each basic region of the protein bindsas an alpha-helix to the two CAAC/T half-sites in the generallypalindromic recognition sequences.

In order to explain the 2:1 stoichiometry for binding Family A consensus22-mer sequence, two TCD dimers must be capable of orienting on theduplex. Note that the Family A 22-mer contains the consensus sequence:(SEQ ID No.: 7) 5′ dGGGCC C GCTGT ACATG C ACACG (SEQ ID No.: 16)   CCCGG G CGACA TGTAC G TGTGCd 5′.There are three very similar sequences shown above in bold: 5′ACAGC, 5′ACATG, 5′ACACG, which were being selected even in rounds 7-10, andremarkably, in the same orientation and order shown above (Table 1).This degree of conservation suggests that one TCD dimer can bind witheach basic recognition helix interacting with each of the 5 nucleotidesequences (containing ACA.TGT triplets) shown in bold. The second TCDdimer may bind to one of the two basic recognition helices recognizingthe 3rd 5 nucleotide sequence in bold.

The six N-terminal residues of TCD are required for binding to theNF-IL6 consensus sequence (Brasier et al. J. Biol. Chem. (1994) 269:1034). Although not part of the basic domain of the protein, the NMRstructural studies conducted by the present inventors demonstrated thatthere is considerable helical content in this region.

E. Nuclease Resistance of Thiolated Aptamers

The sensitivity of the duplexes to DNase I degradation was monitored bynative PAGE. Reaction mixtures contained either 40.5 or 33.8 μg/mlduplex (phosphoryl or thiophosphoryl, respectively) in 205 μL of 50 mMTris, 10 mM MgCl₂, 50 μg/mL BSA, pH 7.5 buffer and 0.12 μg/mL DNase I(Sigma). Aliquots (20 μl) were removed at specific time points and thehydrolysis quenched by adding 4 μl 0.5 M EDTA, 20 μl 90% formamide,followed by boiling and storing at 0° C. Gels were scanned and the66-mer lane integrated using an Image ID gel scanner (Pharmacia).

As shown in FIG. 2, thiophosphorylation of the Family A 66-mer at onlythe dA sites (excepting the primers) results in a duplex that is moreresistant to DNase I degradation than the unmodified 66-mer. Increasednucelase resistance was found for both endonucleases such as DNase I orexonucleases such as Bal 31.

EXAMPLE 2 Specificity of NF-κB Monothioate Aptamers

A. Aptamers

An oligonucleotide duplex of the sequence 5′-CCAG GAGA TTCC AC CCAG GAGATTCC AC CCAG GAGA TTCCAC-3′, termed CK-1 (SEQ ID NO.: 16), wasidentified by Sharma, et al. (Anticancer Res. (1996) 16:61), to be anefficient NF-κB binding aptamer. The original phosphodiester CK-1 duplexsequence contains 3 tandem repeats of a 14-mer NF-κB binding sequence(5′-CCA GGA GAT TCC AC 3′, a.k.a., CK-14 and having SEQ ID NO.: 17). TheCK-1 42-mer duplex oligonucleotide is said to represent the NF-κBbinding site in the G-CSF and GM-CSF promoter to which RelA but not thep50 homodimer binds. The CK-1 decoy ODN has been shown to decrease theexpression of cytokine and immunoglobulin genes in cultured mousesplenocytes. (Khaled, et al., Clinical Immunology & Immunopathology(1998) 86:170). It was argued that CK-1 specifically targeted theactivators of NF-κB regulated gene expression, p50/c-Rel or RelA dimers,and not the repressive p50 homodimers.

It is unlikely, however, that unmodified or phosphodiester ODNs may beuseful as therapeutics because of their short half-life in cells andserum. Phosphorothioate and dithioate internucleotide linkages aretherefore needed. Presumably for this reason Sharma, et al. (AnticancerRes. (1996) 16:61), also reported inhibition of NF-κB in cell cultureusing fully thiolated [S]-ODN duplex decoys with the NF-κB bindingconsensus-like sequence (GGGGACTTCC SEQ ID NO.: NO.: 18).

To determine the effect of monothiolation of the CK-14 sequence on NF-κBbinding, the present inventors chemically synthesized a monothiolatedCK-14 sequence by sulfur oxidation with phosphoramidite chemistry, thesame method used by Sharma to generate the [S]-(GGGGACTTCC) duplex.Using this method, the monothiolated ODN contain in principle 2⁸² or10²⁴ different stereoisomers.

B. Binding of Monothiolated ODN to Various NF-κB/Rel Dimers.

The present applicants used recombinant protein homodimers of p50, p65,and c-Rel showing that the phosphodiester CK-1 sequence could bind toand compete for binding to p65 homodimer, but not p50/p50, in standardelectrophoretic mobility shift assays (EMSA), confirming the publishedresults. (Sharma, et al., Anticancer Res. (1996) 16: 61).

CK-1 did bind and compete for binding to c-Rel. Oligonucleotidescontaining only one copy of the binding site in either a 14-mer (5′-CCAGGA GAT TCC AC; CK-14) or a 22-mer duplex ODN (an IgκB site) behavedsimilarly to the longer version, and served as the first target for thesynthesis of various hybrid backbone-modified aptamers.

FIG. 5 is a graph showing the binding of duplex ODNs using Sharma'steachings and demonstrating that the phosphodiester of CK-1 binds onlyp65/p65 [FIG. 5(A)] and not p50 homodimer. In standard competitivebinding assays, ³²P-IgκB promoter element ODN duplex was incubated withrecombinant p50 or p65 and competitor oligonucleotide (A) phosphodiesterCK-1; (B) phosphorothioate CK-1. The reactions were then run on anondenaturing polyacrylamide gel, and the amount of radioactivity boundto protein and shifted in the gel was quantitated by direct counting.When fully thiosubstituted, the phosphorothioate CK-1 aptamer [FIG.5(B)] equally inhibited p65/p65 and p50/p50. It is the recognition that[S]-ODNs with large numbers of phosphorothioate linkages are “sticky”and tend to bind with poor specificity to proteins that led to one ofthe embodiments of the present invention. Using the method disclosedherein it was determined that if the number of phosphorothioate linkagesis reduced to only 2-4, specificity can be restored, but binding is notenhanced. The original published results of Sharma describe only thespecificity of the phosphodiester oligonucleotides and do not addressthe problem of altered specificity of the phosphorothioates.

EXAMPLE 3 Thioselection of Phosphorodithioate Aptamers Binding to NF-κB

A. Library Generation

A random combinatorial library of normal phosphoryl backboneoligonucleotides was synthesized by an automated DNA synthesizer thatwas programmed to include all 4 monomer bases of the oligonucleotideduring the coupling of residues in a randomized segment. A 62-mer hasbeen constructed with a 22 base pair random central segment flanked by19 and 21 base pair PCR primer regions:5′dATGCTTCCACGAGCCTTTC(N₂₂)CTGCGAGGCGGTAGTCTATTC3′ (SEQ ID NO.: 19). Theresulting library thus exists as a population with potentially 4²²(10¹³) different possible sequences.

B. Thiophosphate Substitution and Selection

The duplex oligonucleotide library with phosphoromonothioate backbonesubstituted at dA positions was then synthesized by PCR amplification ofthe 62-mer template using commercially available Taq polymerase andusing a mix of dATP(αS), dTTP, dGTP and dCTP as substrates. As will beappreciated by those of skill in the art any of the nucleotides may bethe one or more nucleotides that is selected to have the thiolmodification.

The random library generated thereby was screened to identify sequencesthat have affinity to the p65 homodimer. PCR amplification of the singlestranded library provides chiral duplex phosphorothioate 62-mer at alldA positions other than the primers. This material was then incubatedwith the p65 dimer for 10 minutes at 25° C. and filtered throughpre-soaked Millipore HAWP25 mm nitrocellulose filters. The combinatorialthiophosphate duplex library was successfully screened for binding tothe p65 dimer. The filter binding method was modified to minimizenon-specific binding of the thiophosphate oligonucleotides to thenitrocellulose filter.

The thiophosphate substituted DNA was be eluted from the filter underhigh salt and under protein denaturing conditions. Subsequent ethanolprecipitation and another PCR thiophosphate amplification provideproduct pools for additional rounds of selection. In order to increasethe stringency of binding of the remaining pool of DNA in the libraryand select tighter binding members of the library, the KCl concentrationwas increased in subsequent rounds from 50 to 200 mM. The stringency ofselection was also manipulated by increasing the volume of washingsolution as the number of iterations are increased. A negative controlwithout protein was performed simultaneously to monitor any non-specificbinding of the thiophosphate DNA library to the nitrocellulose filter.

Thioselection against the p65•p65 of NF-κB was carried through 10rounds. Cloning and sequencing according to standard methods known tothose in the art was performed after 10 iterations had been completed.From these rounds of selection eight (8) sequences, shown here as theduplex sequence, were obtained: (SEQ ID NO.: 20) 1) 5′GGG GCG GGG GGATAT GGA CAC C3′ 3′CCC CTC CCC CCT ATA CCT GTG G5′ (SEQ ID NO.: 21) 2)5′GGG CTG GTG TGG TAG ACT CCC C3′ 3′CCC GAC CAC ACC ATC TGA GGG G5′ (SEQID NO.: 22) 3) 5′CCC GCC CAC ACA CAC CGC CCC C3′ 3′GGG CGG CTG TGT GTGGCG GGG G5′ (SEQ ID NO.: 23) 4) 5′GGG CCG GGA GAG AAC ATA GCG AC3′ 3′CCCGGC CCT CTC TTG TAT CGC TG5′ (SEQ ID NO.: 24) 5) 5′CCC NCN NNC ACA CACCGC CCC C3′ 3′GGG NGN NNG TGT GTG GCG GGG G5′ (SEQ ID NO.: 25) 6) 5′GGTATA CTC TCC GCC CCT CCC C3′ 3′CCA TAT GAG AGG CGG GGA GGG G5′ (SEQ IDNO.: 26) 7) 5′CCC ACA TGT ACA CGC CGC CCC CGC CC3′ 3′GGG TGT ACA TGT GCGGCG GGG GCG GG5′ (SEQ ID NO.: 27) 8) 5′CCC ACA TGN ACA CNC CGC CCC C3′3′GGG TGT ACN TGT GNG GCG GGG G5′

The sequences were lined up by either their 5′-3′ or 3′-5′ ends choosingthe G rich strand, thus finding a consensus pattern in the sequences.The sequence obtained for a 22-nucleotide variable region in which alldAs were thiolated, which shows somewhat of a conserved consensus sitecontaining two tandem decameric κB motifs separated by G*. A generalconsensus site for the 22-nt variable region of a new combinatoriallibrary was identified which binds tightly to NF-κB:

GGGCG T ATAT G* TGTG GCGGG GG (SEQ ID NO.: 28). Surprisingly, thissequence differs from the CK-1 sequence of 14 bases. The GGGCG isconserved at both ends of the sequence and finishes with a purinepyrimidine alternation of bases (ATAT or GTGT) centered around the G*.The binding characterisitcs of this 22-mer suggests that two p65homodimers bind to the selected sequence and that the p65 homodimersinteract in a head to head fashion enhancing their affinity to themutated DNA.

A binding study done with the sequences from round 10 by ³²P labelingEMSA showed specific binding of the thiolated DNA to the p65 homodimerprotein. Thus, as with the NF-IL6 thioaptamer, the present thiophosphatecombinatorial selection technology achieved selection of a tight bindingaptamer with a sequence that differs from the normal phosphate backboneaptamer selected sequence. Furthermore, the NF-κB thioaptamer exhibitsan approximate two-fold, head-to-head symmetry (assuming A, G=Pu in thecentral 9 bps) centered around G* in the combinatorially selectedsequence. This is similar to the NF-IL6 thioselection aptamer, describedhereinabove, in which high selection constancy was obtained throughoutthe full 22-nucleotide variable region, and the stoichiometry indicatedthat two NF-IL6 bZIP dimers bound per aptamer.

As it appears that two NF-κB dimers bind to the thioselected [S]-ODN,this creates a novel invention providing for the development of evenmore highly selective thiolated aptamers targeted to specific NF-κB/Relhomo- and hetero-dimers, based not only on the protein-DNA contacts, butalso on protein-protein contacts. Orientation of each of the NF-κB/Reldimers on such an aptamer will tightly constrain the optimal dimer-dimercontacts and will presumably will differ for each homo- or hetero-dimer.The present invention provides a thioselection methodology that targetsany number of different protein-protein complexes, not just those fromNF-IL6 and NF-κB/Rel.

Single stranded phosphorothioate selection may be performed in exactlythe same manner as the duplex version. The difference will be the methodof PCR performed during each iteration. Briefly, one primer in the setof 2 must be 5′ biotinylated. Following PCR, the duplex is denatured andthe 2 strands separated using an avidin affinity column. In this way,only one strand of the library will be sampled and enriched during eachiteration.

EXAMPLE 4 NF-κB Aptamers with Specific Placement of Phosphorodithioates:Synthesis, Purification and Binding to NF-κB

One embodiment of the present invention provides a method of preparationof oligonucleotides containing selected phosphorodithioate linkages freeof phosphoromonothioate impurities. One such preparation involves solidphase synthesis using the nucleoside phosphorothioamide method followedby PCR to generate achiral phorphorothioate oligonucleotides based on aselected sequence. Oligonucleotides essentially free from detectablephosphomonothioate linkages are provided using ion exchangechromatography, e.g., using a Mono Q ion exchange purification column.

Using this method, aptamers targeting NF-κB that contain thymidine3′-O-phosphorodithioates in selected positions of an oligonucleotideduplex were synthesized. The total number of dithiolated phosphates wasoptimized in order to minimize non-specific protein binding whileenhancing specific binding to the protein of interest. Bindingaffinities to NF-κB varied with the number and positions of thedithioate backbone substitutions. An aptamer showing specific binding toa single NF-κB dimer in cell culture extracts was obtained.

In one embodiment, dithiolated [S₂]-ODNs were generated using animproved method of solid phase synthesis based on nucleosidephosphorodithioamidite chemistry. Solid-phase synthesis of aptamerscontaining thymidine 3′-O-phosphorodithioates free ofphosphoromonothioates was obtained as follows.

The 14-mer NF-κB binding sequence (5′-CCA GGA GAT TCC AC 3′ SEQ ID NO.:17) was used as the starting point for design of novel NF-κB specificthioaptamers. As it was determined by the present inventors thatcomplete thioation of the CK-1 or CK-14 aptamer results in loss ofspecificity, it is clear that complete thiolation does not provide aneffective agent capable of specifically binding various NF-κB/Reldimers.

Based on the observation that excess thioation leads to a loss ofspecificity in the interaction between the thiolated phosphates and theprotein, the present inventors selected specific nucelotidesthiosubstitution and synthesized 14-mer duplexes with strategicallyplaced dithioate linkages. These substitutions resulted in verysignificant and surprising effects on the function of the 14-mersequence. They not only have the extreme “stickiness” of the fullythiolated aptamer but also exhibit altered binding specificity. Thepresent inventors further found that when only one or twophosphodithioate linkages are placed in a molecule, theinhibition/binding of the oligonucleotide to recombinant protein issimilar to that of the unsubstituted aptamer.

FIG. 6 is a graph showing the competitive binding of the six XBY oligos.With more substitutions, binding by the oligonucleotide increasesdramatically. In standard competitive binding assays, ³²P-IgκB promoterelement ODN was incubated with recombinant p65 and varying amounts ofXBY decoy competitor. The relative binding ability of the unlabeled ODNswas determined by the concentration needed to effectively compete withthe standard labeled ODN. XBY 1 through 6 correspond to CK-14 aptamerswith 1 though 6 dithiophosphate substitutions, respectively.

A. Synthesis of Thymidine 3′-O-phosphorodithioates

The thymidine 3′-O-phosphodiesters of Scheme 1:1 and its complementarysequence of 5′-GTGG AATC TCCTGG-3′ (SEQ ID NO.: 29) were replaced withthymidine 3′-O-phosphorodithioates in two or four positions. Thenucleoside phosphorothioamidite approach was used to synthesize the[S₂]ODNS according to the method of Wiesler, et al., J. Org. Chem.(1996) 61:4272. Using this method, thymidine S-(β-thiobenzoylethyl)pyrrolidinophosphorothioamidite was prepared in ca. 80% yield. Thepurity (ca. 80%) was assayed via ³¹P NMR (δ³¹ P 161.1, 164.7 CD₂Cl₂). Ininitial determinations, oligonucleotide Scheme 1:A(2) (SEQ ID NO.: 40)containing two thymidine 3′-O-phosphorodithioate linkages was firstprepared on a Gene Assembler Plus (Pharmacia) (1.3 mmole).

A modification of the normal coupling cycle for the phosphorothioamiditeDMT yielded a coupling efficiency of ca. 98-99%. Briefly, sulfurizationwas carried out with ³H-1,2-benzodithiole-3-one, 1,1-dioxide (BeaucageReagent), resulting in a normal phosphoramidite DMT-efficiency of ca.99%. The crude oligonucleoside phosphorodithioate (DMT on) was cleavedfrom the support and deblocked by treatment with 28-30% aqueous ammonia(ca. 1.5 ml) in a tightly stoppered vial at 55° C. for 16 hours. Afterremoval of the support, the ammonia solution was concentrated andsubjected to ³¹P NMR analysis, which showed the correct ratio ofphosphorodithioate linkages (δ³¹P ca. 113 ppm) to phosphate linkages(δ³¹ P ca. 0 ppm). ³¹P NMR analysis of this oligonucleotide, however,also showed some small amounts of nucleoside phosphoromonothioates (δ³¹P ca. 58 ppm) as a previously noted contaminate of the procedure.(Okruszek, et al., J. Org. Chem. (1995) 60:6998; Wiesler, et al., J.Org. Chem. (1996) 61:4272.)

The 5′ O-DMT-oligonucleotide Scheme 1:A(2) (SEQ ID NO.: 30) was purifiedby reverse-phase HPLC (Hamilton PRP-1 column), desired fractions werecollected and evaporated. Detritylation was accomplished with 75% aceticacid for 15 min at 0° C. After three diethyl ether extractions, thesolution was neutralized with aqueous ammonia, followed bylyophilization. ³¹P NMR showed increased amounts ofphosphoromonothioates, suggesting that the deprotection step leads tosome desulfurization. If the final 5′-O-DMT protecting group was removedon the synthesizer while still on the solid support, however,desulfurization was diminished.

B. Purification of Thymidine 3′-O-phosphorodithioates

The crude oligonucleotides were dissolved in about 1.0 ml of water, andpurified by FPLC ion exchange (Pharmacia Mono Q 5/5) chromatographyusing the following gradient for purification/time: 0-80 min, 0-100 B %;80-85 min, 100 B %; 85-89 min, 100-0 B % and the following mobile phasesolvents: A) 25 mM Tris-HCl, 1 mM EDTA, pH 8; and B) 25 mM Tris-HCl, 1mM EDTA, 1.0 M NaCl, pH 8. ³¹P NMR showed that the oligonucleotidephosphoromonothioate impurities were not present indicating that Mono Qion exchange column chromatography is able to remove these impurities.

C. Generation of Preferred Substituted Oligonucleotides

The above methodology was applied to the design and preparation ofcomplementary sequences containing two or four thymidine3′-O-phosphorodithioate linkages. The purity of each oligomer (Scheme1:A(3-5): SEQ ID NO.: 31-33) was assayed by ³¹P NMR.

FIG. 8 shows a representative ³¹P NMR spectrum of an oligonucleotideScheme 1:A(5) (SEQ ID NO.: 33) containing four 3′-O-phosphorodithioatelinkages showing the absence of any phosphoromonothioate linkages. The³¹P NMR spectrum of 5 was recorded on a Varian Unity plus spectrometeroperating at 242 MHz. The sample contained ca. 400D A₂₆₀ units dissolvedin 500 μl of D₂O. Chemical shifts were referenced to 85% phosphoricacid. The molecular structure of oligonucleotide 5 was further confirmedby MALDI-MS, the calculated molecular weight is 4422.92 and themolecular weight observed was 4426.13. Peaks corresponding to M-16 orM-32 were not observed confirming the absence of significant quantitiesof oligomers with phosphoromonothioate linkages. Scheme 1. A: SingleStrands: Single-stranded oligonucleotides 1-5 synthesized are shown, inwhich thymidine 3′-O-phosphorodithioate was incorporated in two or fourpositions.  1. 5′-CCAGGAGATTCCAC-3′ SEQ ID NO.: 17  2.5′-CCAGGAGAT_(S2)T_(S2)CCAC-3′ SEQ ID NO.: 30  3.5′-GT_(S2)GGAATCTCCT_(S2)GG-3′ SEQ ID NO.: 31  4.5′-GTGGAAT_(S2)CT_(S2)CCTGG-3′ SEQ ID NO.: 32  5.5′-GT_(S2)GGAAT_(S2)CT_(S2)CCT_(S2)GG-3′ SEQ ID NO.: 33 Scheme 1. B.Duplexes: Duplex aptamers were annealed at 15.75 μM of each strand in 10mM Tris-HCl pH 7.5, 2 mm MgCl₂, 50 mM NaCl, 1mM EDTA.  6. 5′-CCAGGAGATTCCAC-3′ SEQ ID NO.: 34 3′-GG_(S2)TCCTCTAAGG_(S2)TG-5′  7.5′-CCAGGAGATTCCAC-3′ SEQ ID NO.: 35 3′-GGTCC_(S2)TC_(S2)TAAGGTG-5′  8.5′-CCAGGAGATTCCAC-3′ SEQ ID NO.: 363′-GG_(S2)TCC_(S2)TC_(S2)TAAGG_(S2)TG-5′  9.5′-CCAGGAGAT_(S2)T_(S2)CCAC-3 SEQ ID NO.: 373′-GG_(S2)TCCTCTAAGG_(S2)TG-5′ 10. 5′-CCAGGAGAT_(S2)T_(S2)CCAC-3′ SEQ IDNO.: 38 3′-GGTCC_(S2)TC_(S2)TAAGGTG-5′ 11.5′-CCAGGAGAT_(S2)T_(S2)CCAC-3′ SEQ ID NO: 393′-GG_(S2)TCC_(S2)TC_(S2)TAAGG_(S2)TG-5′D. Binding Specificity of the Thiolated Aptamers

In an alternate embodiment, duplex [S₂]-ODN aptamers Scheme 1:B(6-11)(SEQ ID NO.: 34-39) were prepared and their binding to NF-κB wasanalyzed. In standard competitive binding assays as shown in FIG. 9, ³¹Pend-labeled IgκB promoter element oligonucleotide (5′-AGTT GAGG GGACTTTC CCAG GC-3′; SEQ ID NO.: 40) was incubated with recombinant p65²⁸and varying amounts of competitor aptamers Scheme 1:B(6-11). Therelative binding affinity of the unlabeled aptamers was determined bythe concentration needed to effectively compete with the standardlabeled aptamer. When only one strand of the aptamers contain thymidine3′-O-phosphorodithioate Scheme 1:B(6-8), the inhibition/binding of theaptamer to protein was similar to that of the unsubstituted aptamer.With increased dithioate substitution on both strands, binding by the[S₂]-ODNs increased dramatically. FIG. 9 shows the inhibition of p65homodimer binding by dithioate substituted aptamers Scheme 1:B(6-11).

One aptamer of the present invention, duplex aptamer Scheme 1:B(11), SEQID NO.:39 (also termed XBY-6 herein), contains six dithioate linkages onthe two strands. It was found that unlike the fully monothio-substitutedaptamer [S]-ODN CK-14, the present dithiolated [S₂] hybrid backbone ODNXBY-6 binds more tightly to recombinant p65/p65 (5 to 15 fold) than tothe recombinant p50 homodimer in vitro. Significantly, the XBY-6 aptameralso binds to a single NF-κB dimer in cell extracts, while the standardunmodified phosphodiester ODN showed no NF-κB-specific binding inextracts.

The following studies demonstrate the NF-κB subunit specificity ofaptamer binding. Thymidine 3′-O-phosphorodithioate duplex aptamer Scheme1:B(11) (SEQ ID NO.: 39) was incubated with radiolabeled oligonucleotidewith 7OZ/3 cell nuclear extract in the presence or absence of anti-p50antibody. Protein bound ODN duplex was separated on a non-denaturinggel.

It was found that XBY-6 shifts only one complex in nuclear extracts from70Z/3 cells. By using specific antibodies to supershift the complex, p50was identified as one component of the complex. Since XBY-6 binds morepoorly to p50/p50 (by a factor of 5-15-fold) than p50/p65, the shiftedband is unlikely to represent the p50 homodimer. Although this band doesnot co-migrate with either the p50/p50 or p50/p65 bands, the alteredchemical structure changes the mobility of the ODN. Only one major bandwas seen, however, even though the lysate contains at least two majordistinguishable NF-κB complexes (p50 homodimers and p50/p65heterodimers) (data not shown). By binding to p50 but not p65, it isclear that the thioselected aptamer of the present invention candistinguish among various NF-κB dimers within the cell.

EXAMPLE 5 Split Synthesis Combinatorial Chemistry

A. Library Generation

A split synthesis combinatorial chemistry method was developed to createa combinatorial library of [S₂]-ODN agents. In this procedure eachunique member of the combinatorial library is attached to a separatesupport bead. Proteins that bind tightly to only a few of the 10⁴-10⁶different support beads can be selected by deprotecting a single aptamerbead in a 96-well plate in a high-throughput assay, or by binding theprotein directly to the beads and then identifying which beads havebound protein by immunostaining techniques.

To introduce many copies of a single, chemically pure [S₂]-ODN onto eachbead, a “mix and separate” method was used. Applying this method toNF-κB, the XBY-6 sequence was synthesized as a single-strand 33-merhairpin containing a TTTT loop as follows: (SEQ ID NO.: 40)5′dCCAGGAGAT*TCCACTT-TTGT*GGAATCTCCTGGA T* is randomized as either aphos- phorodithioate or a normal phosphate ester.

This was achieved by modifying the normal solid-phase synthesis of the33-mer. On a two column DNA synthesizer, a different thiophosphoramiditewas first added onto both identical supports (at the appropriatesequence position) on each column. After the normal cycle of S oxidationand blocking (which introduces the dithiophosphate linkage at thisposition), the support beads were removed from the columns, mixedtogether and the mixture reintroduced into both columns. At the nextrandomized position (the second T in the above sequence), athiophosphoramidite with either a different or the same base (T in theexample cited) was then added to each of the columns. Cycles of mixingand separating may be continued for n internucleoside dithiophosphates(n=2 in the present example). The dithiophosphate-modifiedoligonucleotide was deblocked and either removed from the column (andpurified) or retained on the column.

B. NF-κB Selection

Target protein is bound to the beads and washed at various salt and ureaconcentrations to remove weakly bound proteins. Support beads that bindthe protein may be visualized under a microscope by adding animmunostaining agent targeted to the protein, and physically separatedfrom the unstained (unbound) beads. Alternatively, multiwavelength flowcytometry and cell sorting can be used for visualization and sorting ofthe protein-bound aptamer beads. As another alternative, high-throughputscreening of the purified aptamers (one from each bead) can be used inn-well selection plates.

C. Sequencing

After selection, the site of dithiophosphate modification may beidentified for the separated aptamer (or covalently linked aptamerreleased from the bead). The difference in chemical reactivity betweenphosphate and phosphorothioate and dithioates is exploited to directlysequence the aptamer and locate the thiolated internucleoside linkagesindependent of the base sequence. Briefly, after ³²P-end labeling, thehybrid [S₂]-ODNs are alkylated with agents such as 2-iodoethanol, whilenormal phosphates are not. Addition of dilute NaOH cleaves only at thethio-(or dithio-)phosphate. Standard sequencing gel electrophoreticmethods are used to determine the size of the cleaved fragments, andthus the position of the modified phosphate backbone (Gish, G. &Eckstein, F., Science (1988)240:1520).

When sequencing of the dithioates was conducted using this technique,reaction of O,O-diethyldithiophosphate with iodoethanol gavequantitatively dithiophosphate triester, which was stable under thecondition of alkylation. With the addition of dilute sodium hydroxide,the triester was hydrolyzed to O,O-diethyl phosphate. When thed[T_(S2)T] was treated with 2-iodoethanol, however, direct conversion toTpT, thymidine 5′-O-monophosphate and thymidine 3′-O-monophosphate wereassayed by ³¹P NMR. TpT was identical to the authentic sample bycomparing their retention time on RP-HPLC chromatography. The differencein reactivity between T_(PS2)T and 11-mer oligonucleotide containing asingle monothioate linkage was observed.

D. Separation by Anion-Exchange Chromatography on a Mono Q Column

A variety of separation strategies have been used for the analysis andpurification of synthetic phosphorodithioate oligonucleotides. Theselection of the appropriate separation method is dictated primarily bythe oligonucleotide purity requirements of the application. Sincechemical by-products generated in the synthesis of phosphorodithioatesmay be toxic to cells in tissue culture or in vivo, the chemicalauthenticity of a particular oligonucleotide phosphorodithioate may becrucial if the oligonucleotide is to be administered in humans as adrug. The ability to purify oligonucleotide phosphorodithioates on aroutine basis is essential.

Purification of oligonucleotide phosphorodithioates bearing less than50% dithioate linkages by ion-exchange HPLC has been mentioned. Neitherthe separation of synthetic phosphorodithioate oligonucleotides fromphosphoromonothioate impurities by anion-exchange chromatography nordithio-dependent separation has been previously achieved. Therefore,provided herein is a new and effective method of ion-exchange FPLCseparation of synthetic phosphorodithioate oligonucleotides fromphosphoromonothioate contaminants by anion-exchange chromatography on aMono Q column.

Oligodeoxyribonucleotides of the base sequence 5′-CCAGGAGATTCCAC-3′ (SEQID NO.: 17) containing either phosphodiester internucleotide linkages ortwo phosphoromonothioate or two phosphorodithioate internucleotidelinkages were synthesized and analyzed using the same gradient on a MonoQ column. The retention time of the phosphodiester oligonucleotide was43.8 min, compared to 47.1 min for the phosphoromonothioate and 56.8 minfor the phosphorodithioate oligonucleotides. It is clearly evident thatthe phosphodiester, phosphoromonothioate and phosphorodithioate werewell separated.

The retention times of the oligonucleotide phosphorodithioate using aMono Q column and a linear gradient of buffers A and B were listed.

The Mono Q purified oligonucleotide phosphorodithioates below were shownto be absent of any monothioate contamination by ³¹P NMR. FPLC Buffer A:25 mM Tris-HCl, 1 mM EDTA, pH = 8 Buffer B: 25 mM Tris-HCl, 1 mM EDTA, 1M NaCl, pH = 8 Condition: Time B % 0.0 0.0 80.0 100 84.20 100 85.40 0.089.0 0.0 90.0 0.0 (SEQ ID NO.: 17) A025: 5′-CCAG GA GA TTCC AC-3′ Rt =43.086 (SEQ ID NO.: 42) A025: 5′-CCAG GA GA T_(S)T_(S)CC AC-3′ Rt =46.156, 47.125, 48.313 (SEQ ID NO.: 32) A036:5′-GTGGAAT_(S2)CT_(S2)CCTGG-3′ Rt = 56.905 (SEQ ID NO.: 31) A037:5′-GT_(S2)GGAATCTCCT_(S2)GG-3′ Rt = 57.870 (SEQ ID NO.: 30) B37: 5′-CCAGGA GA T_(S2)T_(S2)CC AC-3′ Rt = 56.833 (SEQ ID NO.: 43) A070:5′-CCA_(S2)G GA GA TTCC A_(S2)C-3′ Rt = 57.265 (SEQ ID NO.: 44) A071:5′-CCAG GA_(S2) GA_(S2) TTCC AC-3′ Rt = 58.983 (SEQ ID NO.: 45) A072:5′-CCA_(S2)G GA_(S2) GA_(S2) TTCC AC-3′ Rt = 65.738 (SEQ ID NO.: 46)A073: 5′-CCAG GA_(S2) GA_(S2) TTCC A_(S2)C-3′ Rt = 67.791 (SEQ ID NO.:33) B38: 5′-GT_(S2)GGAAT_(S2)CT_(S2)CCT_(S2)GG-3′ Rt = 73.326 (SEQ IDNO.: 47) A069: 5′-CCA_(S2)G GA_(S2) GA_(S2) TTCC A_(S2)C-3′ Rt = 75.568(SEQ ID NO.: 48) B51: 5′-CCA_(S2)G _(S2)GA GA T_(S2)T_(S2)CC AC-3′ Rt =77.805

The variation in the retention times of the dithioates supports theobservation by the present inventors that a major contributor to theinherent “stickiness” of the dithioates is the result of the poor cationbinding by the dithioate relative to the normal phosphoryl group in theaptamers. Thus, with increasing numbers of dithioates shown in the abovetable, higher concentrations of NaCl are required to desorb the boundaptamer from the anion-exchange column. Aptamers with two, three or fourdithioates, e.g., show an average retention time of 56 min (ca. 0.7 MNaCl), 66 min (ca. 0.83 M NaCl), and 75 min (0.94 M NaCl), respectively.This retention data provides confirmation that the enhanced affinity ofthe dithioate aptamers is attributable at least in part to electrostaticeffects.

EXAMPLE 6 Bio-Activity of NF-kB Specific Thioaptamers

A. LPS Model

Outbred Hartley guinea pigs (400 gm, male) were inoculatedintraperitoneally with doses of lipopolysaccharide (E. Coli 011:B4).Blood samples collected 2 hours later were assayed for tumor necrosisfactor (TNF) by standard bioassay. FIG. 11 a shows the dose relationshipbetween LPS dose and serum TNF. Each bar represents the mean withstandard deviation of an assay with three animals per group.

B. Effect of Thioaptamer Administration on TNF Response

Guinea pigs were pre-treated with intraperitoneal inoculation of XBY-6(SEQ ID NO.: 39) (50 μg in Tfx-50 liposomes, Promega), or emptyliposomes, 1 hour before challenge with 100 μg/kg LPS or diluent alone(PBS). Serum TNF levels at 2 hours post LPS challenge are shown on FIG.11 b. Each bar represents average of 2 animals per group. The datagraphed in FIGS. 11 a and 11 b demonstrate the in vivo physiologicdeactivation of serum TNF production in mice treated with the XBY-6aptamer developed using the method of the present invention.

While this invention has been described with reference to illustrativeembodiments, this description is not intended to be construed in alimiting sense. Various modifications and combinations of illustrativeembodiments, as well as other embodiments of the invention, will beapparent to persons skilled in the art upon reference to thedescription. It is therefore intended that the appended claims encompasssuch modifications and enhancements.

1-17. (canceled)
 18. An achiral oligonucleotide that specifically bindsa target comprising a sequence of nucleotides one or more nucleotides insequence are thiophosphate modified.
 19. The achiral oligonucleotide ofclaim 18, wherein the one or more thio-modified nucleotide in theachiral oligonucleotide is selected from the group consisting ofdATP(αS), dTTP(αS), dCTP(αS) and dGTP(αS), dATP(S₂), dTTP(S₂), dCTP(S₂)and dGTP(S₂).
 20. The achiral oligonucleotide of claim 18, wherein notall like nucleotides have a thiomodified phosphate group.
 21. Theachiral oligonucleotide of claim 18, wherein only one nucleotide has athiomodified phosphate group.
 22. The achiral oligonucleotide of claim18, wherein two nucleotides have a thiomodified phosphate group.
 23. Anachiral oligonucleotide that specifically binds a target comprising asequence of nucleotides wherein one or more of the nucleotides insequence are dithiophosphates.
 24. The achiral oligonucleotide of claim23, wherein the one or more thio-modified nucleotide in the achiraloligonucleotide is selected from the group consisting of dATP(αS),dTTP(αS), dCTP(αS), dGTP(αS), dATP(S₂), dTTP(S₂), dCTP(S₂) and dGTP(S₂).25. The achiral oligonucleotide of claim 23, wherein not all likenucleotides have a thiomodified phosphate group.
 26. The achiraloligonucleotide of claim 23, wherein only one nucleotide has athiomodified phosphate group.
 27. The achiral oligonucleotide of claim23, wherein two nucleotides have a thiomodified phosphate group.
 28. Theachiral oligonucleotide of claim 23, wherein between approximately 10and 80% of the nucleotides in the sequence are thiomodified. 29-39.(canceled)
 40. The achiral oligonucleotide of claim 18, furthercomprising one or more pharmaceutically acceptable salts.
 41. Theachiral oligonucleotide of claim 18, further comprising a diluent. 42.The achiral oligonucleotide of claim 18, wherein the achiraloligonucleotide binds specifically to an NF-kB/Rel protein
 43. Theachiral oligonucleotide of claim 18, wherein the achiral oligonucleotidebinds specifically to an NF-kB homodimer.
 44. The achiraloligonucleotide of claim 18, wherein the achiral oligonucleotide bindsspecifically to an NF-kB heterodimer.
 45. The achiral oligonucleotide ofclaim 18, wherein the oligonucleotide further comprises a detectablelabel.
 46. The achiral oligonucleotide of claim 18, wherein the achiraloligonucleotide is single stranded.
 47. The achiral oligonucleotide ofclaim 23, further comprising one or more pharmaceutically acceptablesalts.
 48. The achiral oligonucleotide of claim 23, further comprising adiluent.
 49. The achiral oligonucleotide of claim 23, wherein theachiral oligonucleotide binds specifically to an NF-kB/Rel protein 50.The achiral oligonucleotide of claim 23, wherein the achiraloligonucleotide binds specifically to an NF-kB homodimer.
 51. Theachiral oligonucleotide of claim 23, F-kB heterodimer.
 52. The achiraloligonucleotide of claim 23, wherein the aptamer comprises one or morethiomodifications as set forth in SEQ ID NOS: 32, 33 and
 38. 53. Theachiral oligonucleotide of claim 23, wherein the achiral oligonucleotidefurther comprises a detectable label.
 54. The achiral oligonucleotide ofclaim 23, further comprising one or more pharmaceutically acceptablesalts.
 55. The achiral oligonucleotide of claim 23, further comprising adiluent.
 56. The achiral oligonucleotide of claim 23, wherein theachiral oligonucleotide is single stranded.
 57. A partiallythio-modified achiral aptamer that binds to a human NF-κB proteincomplex.